Safe-harbor loci, which allow for robust expression of a transgene integrated into the genome of a cell, provide a defined insertion cite for large exogenous DNA such as mini-gene and reporter cassettes. For example, PPP1R12C/AAVS1 and hRosa26 safe harbors have been used in genome engineering of human pluripotent stem cells by conventional or nuclease-enhanced gene targeting (Trion, S. et al., Nature biotechnology 25, 1477-1482 (2007) and Zou, J. et al., Blood 117, 5561-5572 (2011)). While Zinc Finger Nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and CRISPR (clustered regularly interspaced short palindromic repeat) RNA-guided Cas nuclease (CRISPR/Cas) have been used to show efficient gene editing in pluripotent stem cells (Hockemeyer, D. et al., Nature biotechnology 29, 731-734 (2011); Mali, P. et al., Science 339, 823-826 (2013); Zou, J. et al., Cell Stem Cell 5, 97-110 (2009)), one-step modification of multiple loci in stem cells was only recently demonstrated in mouse embryonic stem cells (ESCs) and embryos by non-homologous end-joining (NHEJ) or homology-directed repair (HDR) (Wang, H. et al., Cell 153, 910-918 (2013) and Yang, H. et al., Cell 154, 1370-1379 (2013)). To date, multiplexed knock-in or transfer of large DNA fragment has not been reported in human pluripotent or multi-potent stem cells, although such engineered human stem cells are highly valuable for multi-lineage labeling, drug-screening, and gene therapy.
Neural stem cells (NSCs) are multipotent, self-renewing cells found in the central nervous system (CNS), capable of differentiating into neurons and glia. NSCs can be obtained from several sources; allogeneic sources include fetal tissue, cadaveric samples, while autologous sources can come from brain biopsies or differentiated induced pluripotent cells (iPSCs). Due to the obtainability of NSCs from fetal tissue, adult brain biopsies, cadavers, and iPSCs, genomic engineering of NSCs will greatly enhance their versatility in therapeutic application.
Neurons in the central and peripheral nervous systems degenerate as part of the normal function of human development and aging. Pathological neuron degeneration, however, is a serious condition seen in several neurological disorders. Neuronal degeneration can be specific or diffuse, and can lead to sensory, motor and cognitive impairments. Neurodegenerative disorders encompass a range of seriously debilitating conditions including Parkinson's disease, amyotrophic lateral sclerosis (ALS, “Lou Gehrig's disease”), multiple sclerosis, Huntington's disease, Alzheimer's disease, Pantothenate kinase associated neurodegeneration (PKAN, formerly Hallervorden-Spatz syndrome), multiple system atrophy, diabetic retinopathy, multi-infarct dementia, macular degeneration, and the like. These conditions are characterized by a gradual but relentless worsening of the patient's condition over time. These disorders affect a large population of humans, especially older adults. Nevertheless, there are limited treatment options for these disorders.
Several strategies are being pursued to develop therapies for neurodegenerative disorders, including Parkinson's disease. For Parkinson's disease, the techniques range from the use of dopaminotrophic factors (Takayama et al., Nature Med. 1:53-58, 1995) and viral vectors (Choi-Lundberg et al., Science 275:838-841, 1997) to the transplantation of primary xenogeneic tissue (Deacon et al., Nature Med. 3:350-353, 1997). Transplantation of dopaminergic neurons is a clinically promising experimental treatment in late stage Parkinson's disease. More than 200 patients have been transplanted worldwide (Olanow et al., Trends Neurosci. 19:102-109, 1996), and clinical improvement has been confirmed (Olanow et al., supra, and Wenning et al., Ann. Neurol. 42:95-107, 1997) and was correlated to good graft survival and innervation of the host striatum (Kordower et al., N. Engl. J. Med. 332:1118-1124, 1995). However, fetal nigral transplantation therapy generally requires human fetal tissue from at least 3-5 embryos to obtain a clinically reliable improvement in the patient. A different source of these neurons is clearly needed.